Melanocortins, working through MC4Rs, regulate energy balance. POMC neurons release the MC4R agonist, ?-MSH, and promote negative energy balance, while AgRP neurons release the antagonist, AgRP, and do the opposite. Consistent with these opposing roles, opto- and chemo-genetic stimulation of POMC neurons causes hypophagia and weight loss, while similar stimulation of AgRP neurons produces hyperphagia and weight gain - with prolonged effects being mediated by AgRP through its action on MC4Rs. Importantly, the ?-MSH/MC4R pathway also operates in humans, as evidenced by marked obesity in individuals lacking either ?-MSH or MC4Rs. Despite the established importance of MC4Rs in both mice and humans, the neural mechanisms by which they regulate energy balance, and in particular hunger/satiety, have been unknown. Addressing this has been a major focus of our studies. Recently, by generating and using Mc4rt2a-Cre/+ mice to map and manipulate MC4R+ neurons, we discovered that a) real-time activation or inhibition of PVHMC4R satiety neurons exerts bidirectional control over feeding, b) that these neurons are monosynaptically and functionally downstream of AgRP neurons (which promote hunger by inhibiting their activity), and finally c) that these neurons induce satiety via direct projections to the lateral parabrachial nucleus (LPBN). Importantly, when caloric deprivation-induced hunger is present, reduction of hunger via direct optogenetic activation of this PVHMC4R ==> LPBN satiety circuit, independent of eating, has positive emotional valence. The satiating and positive affective nature of this PVHMC4R ==> LPBN circuit highlight it as a potential target for anti-obesity drug development. The goal of this grant is to establish the mechanisms and pathways by which this PVHMC4R ==> LPBN satiety circuit prevents hunger. In Aim 1, we will determine the molecular signature of PVHMC4R satiety neurons. Towards these ends, we will employ a number of innovative single neuron RNA-seq strategies. The goals are to create a cellular transcriptional atlas of the PVH (using Drop-seq), determine the unique distinguishing features of PVHMC4R satiety neurons versus all other PVH neurons, and identify new proteins affecting function. In Aim 2 we are using an innovative 1.5 synapse rabies mapping strategy to identify monosynaptic afferents to PVHMC4R satiety neurons. Preliminary studies have discovered that significant input comes from local PVH afferents, in addition to distant afferents. We are using a variety of means to study and transcriptionally identify these afferents. In Aim 3, we are employing a variety of approaches, including single neuron nuclei RNA-seq, to discover the identity of the downstream LPBN neurons engaged by upstream PVHMC4R satiety neurons. Finally, in Aim 4, we will determine the means by which these downstream LPBN satiety neurons decrease hunger. As the LPBN relays 1 information to higher structures including the amygdala, BNST and insular cortex, these studies will likely advance our understanding of how the satiety- promoting, positive affect-inducing PVHMC4R ==> LPBN circuit controls hunger/satiety, and ultimately eating.